Chemotaxis Analysis of RAW264.7 Cells

The chemotaxis analysis was according to our previous studies with some modifications [23 (link)]. A 24-well Transwell plate (8 μm pore size, Corning, NY, USA) was used to measure the chemotactic ability of the RAW274.7 cells. HK-2 cells were added into the lower chamber and transfected with a vector carrying a scrambled or the shVHL [24 (link)] sequence by electroporation. After incubation for 48 hours, the cells were treated with the indicated inhibitors for 24 hours in a serum-free medium supplemented with 5% bovine serum albumin (BIONOVAS, Toronto, Canada). Then, 1 × 10⁵ RAW264.7 cells were added to the upper chamber with an uncoated membrane in a serum-free medium supplemented with 5% bovine serum albumin (BIONOVAS, Toronto, Canada). In separate experiments, RAW264.7 cells were exposed to recombinant human lipocalin 2/NGAL protein (rhLCN2) (Novus Biologicals, CO, USA) added to the serum-free medium and their migration measured. After 24 hours, the migratory cells on the underside of the membrane of the upper chamber were stained with 0.1% crystal violet for 5 minutes, washed with H₂O, and scanned using an EPSON V750 PRO scanner. The cells were destained for crystal violet with methanol for 15 minutes and measured at OD570 with the Synergy HT (BioTek, VT, USA).